2-(aryl or heteroaryl-)phenyl (AZA)benzofuran compounds for the treatment of hepatitis C

ABSTRACT

Compounds of Formula I, including their salts, as well as compositions and methods of using the compounds are set forth. The compounds have activity against hepatitis C virus (HCV) and may be useful in treating those infected with HCV: formula (I).

CROSS REFERENCE TO RELATED APPLICATION

This non-provisional application claims the benefit of U.S. ProvisionalApplication Ser. No. 62/001,243 filed May 21, 2014 which is hereinincorporated by reference in its entirety.

FIELD OF THE INVENTION

The invention relates to novel compounds, including their salts, whichhave activity against hepatitis C virus (HCV) and which are useful intreating those infected with HCV. The invention also relates tocompositions and methods of making and using these compounds.

BACKGROUND OF THE INVENTION

Hepatitis C virus (HCV) is a major human pathogen, infecting anestimated 170 million persons worldwide-roughly five times the numberinfected by human immunodeficiency virus type 1.A substantial fractionof these HCV infected individuals develop serious progressive liverdisease, including cirrhosis and hepatocellular carcinoma (Lauer, G. M.;Walker, B. D. N. Engl. J. Med. 2001, 345, 41-52).

HCV is a positive-stranded RNA virus. Based on a comparison of thededuced amino acid sequence and the extensive similarity in the5′-untranslated region, HCV has been classified as a separate genus inthe Flaviviridae family. All members of the Flaviviridae family haveenveloped virions that contain a positive stranded RNA genome encodingall known virus-specific proteins via translation of a single,uninterrupted, open reading frame.

Considerable heterogeneity is found within the nucleotide and encodedamino acid sequence throughout the HCV genome. At least six majorgenotypes have been characterized, and more than 50 subtypes have beendescribed. The major genotypes of HCV differ in their distributionworldwide, and the clinical significance of the genetic heterogeneity ofHCV remains elusive despite numerous studies of the possible effect ofgenotypes on pathogenesis and therapy.

The single strand HCV RNA genome is approximately 9500 nucleotides inlength and has a single open reading frame (ORF) encoding a single largepolyprotein of about 3000 amino acids. In infected cells, thispolyprotein is cleaved at multiple sites by cellular and viral proteasesto produce the structural and non-structural (NS) proteins. In the caseof HCV, the generation of mature non-structural proteins (NS2, NS3,NS4A, NS4B, NS5A, and NS5B) is effected by two viral proteases. Thefirst one is believed to be a metalloprotease and cleaves at the NS2-NS3junction; the second one is a serine protease contained within theN-terminal region of NS3 (also referred to as NS3 protease) and mediatesall the subsequent cleavages downstream of NS3, both in cis, at theNS3-NS4A cleavage site, and in trans, for the remaining NS4A-NS4B,NS4B-NS5A, NS5A-NS5B sites. The NS4A protein appears to serve multiplefunctions, acting as a cofactor for the NS3 protease and possiblyassisting in the membrane localization of NS3 and other viral replicasecomponents. The complex formation of the NS3 protein with NS4A seemsnecessary to the processing events, enhancing the proteolytic efficiencyat all of the sites. The NS3 protein also exhibits nucleosidetriphosphatase and RNA helicase activities. NS5B (also referred to asHCV polymerase) is a RNA-dependent RNA polymerase that is involved inthe replication of HCV. The HCV NS5B protein is described in “StructuralAnalysis of the Hepatitis C Virus RNA Polymerase in Complex withRibonucleotides (Bressanelli; S. et al., Journal of Virology 2002,3482-3492; and Defrancesco and Rice, Clinics in Liver Disease 2003, 7,211-242.

Currently, the most effective HCV therapy employs a combination ofalpha-interferon and ribavirin, leading to sustained efficacy in 40% ofpatients (Poynard, T. et al. Lancet 1998, 352, 1426-1432). Recentclinical results demonstrate that pegylated alpha-interferon is superiorto unmodified alpha-interferon as monotherapy (Zeuzem, S. et al. N.Engl. J. Med. 2000, 343, 1666-1672). However, even with experimentaltherapeutic regimens involving combinations of pegylatedalpha-interferon and ribavirin, a substantial fraction of patients donot have a sustained reduction in viral load. Thus, there is a clear andimportant need to develop effective therapeutics for treatment of HCVinfection.

HCV-796, an HCV NS5B inhibitor, has shown an ability to reduce HCV RNAlevels in patients. The viral RNA levels decreased transiently and thenrebounded during dosing when treatment was with the compound as a singleagent but levels dropped more robustly when combined with the standardof care which is a form of interferon and ribavirin. The development ofthis compound was suspended due to hepatic toxicity observed duringextended dosing of the combination regimens. U.S. Pat. No. 7,265,152 andthe corresponding PCT patent application WO2004/041201 describecompounds of the HCV-796 class. Other compounds have been disclosed; seefor example, WO2009/101022, as well as WO 2012/058125.

What is therefore needed in the art are additional compounds which arenovel and are effective against hepatitis C. Additionally, thesecompounds should provide advantages for pharmaceutical uses, forexample, with regard to one or more of their mechanism of action,binding, inhibition efficacy, target selectivity, solubility, safetyprofiles, and/or bioavailability. Also needed are new formulations andmethods of treatment which utilize these compounds.

SUMMARY OF THE INVENTION

One aspect of the invention is a compound of Formula I includingpharmaceutically acceptable salts thereof:

wherein

-   m is 0 or 1;-   X is N or C—R¹⁰;-   R¹ is methyl;-   R² is Ar¹;-   Ar¹ is phenyl, 5-membered heteroaryl or 6-membered heteroaryl, and    is substituted with 0-3 substituents selected from the group of    cyano, halo, alkyl, cycloalkyl, haloalkyl, OH, OR¹⁰¹, haloalkoxy,    NH₂, NR¹⁰²R¹⁰³, COOR¹⁰²R¹⁰³, S(O)₂NR¹⁰²R¹⁰³, and NR¹⁰¹CONR¹⁰²R¹⁰³;-   R¹⁰¹ is hydrogen, alkyl or cycloalkyl with 0-3 substituents selected    from halo, hydroxyl, alkoxy, and haloalkoxy;-   R¹⁰², R¹⁰³ are each independently hydrogen, alkyl or cycloalkyl with    0-3 substituents selected from halo, hydroxyl, alkoxy, and    haloalkoxy; or-   R¹⁰² and R¹⁰³ can form a ring by joining two atoms, one from each of    R¹⁰² and R¹⁰³;-   R³ is hydrogen, halo, or alkyl;-   R⁴, R⁵, R⁶, R⁷, R⁸ are each independently selected from the group of    hydrogen, halo, alkyl, cycloalkyl, haloalkyl, halocycloalkyl,    hydroxyalkyl, hydroxycycloalkyl, alkoxyalkyl, alkoxycycloalkyl,    alkoxy, hydroxyalkyloxy, alkoxyalkyloxy, COOR²⁰¹ and    CON(R²⁰²)(R²⁰³);-   R²⁰¹ is hydrogen, alkyl or cycloalkyl with 0-3 substituents selected    from halo, hydroxyl, alkoxy, and haloalkoxy;-   R²⁰², R²⁰³ are each independently selected from the group of    hydrogen, alkyl, cycloalkyl, alkenyl, cycloalkyl alkynyl, cyclic    ether, cyclic amine, lactame, fused bicyclic alkyl, bridged bicyclic    alkyl, spiro bicyclic alkyl, fused bicyclic ether, bridged bicyclic    ether, spiro bicyclic ether, fused bicyclic amine, bridged bicyclic    amine and spiro bicyclic amine, with 0-3 substituents selected from    the group of halo, OH, OR¹⁰⁴, NH₂, NR¹⁰⁵R¹⁰⁶, COOR¹⁰⁴, CONR¹⁰⁵R¹⁰⁶,    S(O)₂R¹⁰⁴, S(O)₂NR¹⁰⁵R¹⁰⁶, NR¹⁰⁴CONR¹⁰⁵R¹⁰⁶,-   OR¹⁰⁴CONR¹⁰⁵R¹⁰⁶, C(═NR¹⁰⁷)NR¹⁰⁵R¹⁰⁶, NR¹⁰⁸C(═NR¹⁰⁷)NR¹⁰⁵R¹⁰⁶,    haloalkoxy, Ar², O—Ar², and NR¹⁰⁵—Ar²; or-   R²⁰² and R²⁰³ can form a ring by joining two atoms, one from each of    R²⁰² and R²⁰³;-   R²⁰² and R²⁰³ can also form bicyclic or tricyclic rings by joining    multiple atoms from each of R²⁰² and R²⁰³;-   R¹⁰⁴ is hydrogen, alkyl or cycloalkyl with 0-3 substituents selected    from halo, hydroxyl, alkoxy, and haloalkoxy;-   R¹⁰⁵, R¹⁰⁶ are each independently hydrogen, alkyl or cycloalkyl with    0-3 substituents selected from halo, hydroxyl, alkoxy, and    haloalkoxy; or-   R¹⁰⁵R¹⁰⁶ can form a ring by joining two atoms, one from each of R¹⁰⁵    and R¹⁰⁶;-   R¹⁰⁷, R¹⁰⁸ are each independently hydrogen, alkyl or cycloalkyl with    0-3 substituents selected from halo, hydroxyl, alkoxy, and    haloalkoxy; or-   R¹⁰⁷ and R¹⁰⁸ can form a ring by joining two atoms, one from each of    R¹⁰⁷ and R¹⁰⁸;-   Ar² is phenyl, 5-membered heteroaryl or 6-membered heteroaryl, and    is substituted with 0-3 substituents selected from the group of    cyano, halo, alkyl, cycloalkyl, haloalkyl, OH, OR²⁰⁴, haloalkoxy,    NH₂, NR²⁰⁵R²⁰⁶, COOR²⁰⁴, CONR²⁰⁵R²⁰⁶, S(O)₂R²⁰⁴, S(O)₂NR²⁰⁵R²⁰⁶, and    NR²⁰⁴CONR²⁰⁵R²⁰⁶;-   R²⁰⁴ is hydrogen, alkyl or cycloalkyl with 0-3 substituents selected    from halo, hydroxyl, alkoxy, and haloalkoxy;-   R²⁰⁵, R²⁰⁶ are each independently hydrogen, alkyl or cycloalkyl with    0-3 substituents selected from halo, hydroxyl, alkoxy, and    haloalkoxy; or-   R²⁰⁵ and R²⁰⁶ can form a ring by joining two atoms, one from each of    R²⁰⁵ and R²⁰⁶;-   R⁹ is selected from the group of hydrogen, halo, alkyl, haloalkyl,    cycloalkyl, alkoxy, Ar³ and NR³⁰¹R³⁰²;-   R³⁰¹ is selected from the group of hydrogen, alkyl, cycloalkyl,    (cycloalkyl)alkyl, benzyl, alkylcarbonyl, haloalkyl carbonyl, phenyl    carbonyl, (alkoxyphenyl)carbonyl, alkylsulfonyl, phenylsulfonyl,    (alkoxyphenyl)sulfonyl and (haloalkoxyphenyl)sulfonyl;-   R³⁰² is hydrogen, alkyl, hydroxyalkyl, or alkoxyalkyl; or-   R³⁰¹ and R³⁰² taken together with the nitrogen to which they are    attached is oxazolidinonyl or dioxothiazinyl;-   Ar³ is phenyl, 5-membered heteroaryl or 6-membered heteroaryl, and    is substituted with 0-3 substituents selected from cyano, halo,    alkyl, cycloalkyl, haloalkyl, OH, OR³⁰³, haloalkoxy, NH₂, NR³⁰⁴R³⁰⁵,    COOR³⁰³, CONR³⁰⁴R³⁰⁵, S(O)₂R³⁰³, S(O)₂NR³⁰⁴R³⁰⁵, and    NR³⁰³CONR³⁰⁴R³⁰⁵;-   R³⁰³ is hydrogen, alkyl or cycloalkyl with 0-3 substituents selected    from halo, hydroxyl, alkoxy, and haloalkoxy;-   R³⁰⁴, R³⁰⁵ are each independently hydrogen, alkyl or cycloalkyl with    0-3 substituents selected from halo, hydroxyl, alkoxy, and    haloalkoxy; or-   R³⁰⁴ and R³⁰⁵ can form a ring by joining two atoms, one from each of    R³⁰⁴ and R³⁰⁵;-   R¹⁰ is hydrogen, alkyl, halo, N(R⁴⁰¹)(R⁴⁰²), or alkylsulfonyl;-   R⁴⁰¹ and R⁴⁰² are each independently selected from the group of    hydrogen, alkyl, hydroxyalkyl, alkoxyalkyl, alkylsulfonyl, and    alkylsulfonylalkyl;-   or N(R⁴⁰¹)(R⁴⁰²) taken together is azetidinyl, pyrrolidinyl,    piperidinyl, or piperazinyl, and is substituted with 0-2    substituents selected from alkyl, hydroxyalkyl, and hydroxy.

The invention also relates to pharmaceutical compositions comprising acompound of Formula I, including a pharmaceutically acceptable saltthereof, and a pharmaceutically acceptable carrier, excipient and/ordiluent.

In addition, the invention provides one or more methods of treatinghepatitis C infection comprising administering a therapeuticallyeffective amount of a compound of Formula I to a patient.

Also provided as part of the invention are one or more methods formaking the compounds of Formula I.

The present invention is directed to these, as well as other importantends, hereinafter described.

DETAILED DESCRIPTION OF THE EMBODIMENTS

As used herein, the singular forms “a”, “an”, and “the” include pluralreference unless the context specifically dictates otherwise.

Unless otherwise specifically set forth elsewhere in the application,the following terms may be used herein and shall have the followingmeanings: “Hydrogen” or “H” refers to hydrogen, including its isotopes,such as deuterium. “Halo” means fluoro, chloro, bromo, or iodo. “Alkyl”means a straight or branched alkyl group composed of 1 to 6 carbons.“Alkenyl” means a straight or branched alkyl group composed of 2 to 6carbons with at least one double bond. “Cycloalkyl” means a monocyclicring system composed of 3 to 7 carbons. “Hydroxyalkyl,” “alkoxy” andother terms with a substituted alkyl moiety include straight andbranched isomers composed of 1 to 6 carbon atoms for the alkyl moiety.“Halo” includes all halogenated isomers from monohalo substituted toperhalo substituted in substituents defined with halo, for example,“Haloalkyl” and “haloalkoxy”, “halophenyl”, “halophenoxy.” “Aryl” meansa monocyclic or bicyclic aromatic hydrocarbon groups having 6 to 12carbon atoms, or a bicyclic fused ring system wherein one or both of therings is a phenyl group. Bicyclic fused ring systems consist of a phenylgroup fused to a four- to six-membered aromatic or non-aromaticcarbocyclic ring. Representative examples of aryl groups include, butare not limited to, indanyl, indenyl, naphthyl, phenyl, andtetrahydronaphthyl. “Heteroaryl” means a 5 to 7 membered monocyclic or 8to 11 membered bicyclic aromatic ring system with 1-5 heteroatomsindependently selected from nitrogen, oxygen, and sulfur. Parentheticand multiparenthetic terms are intended to clarify bonding relationshipsto those skilled in the art. For example, a term such as ((R)alkyl)means an alkyl substituent further substituted with the substituent R.Substituents which are illustrated by chemical drawing to bond atvariable positions on a multiple ring system (for example a bicyclicring system) are intended to bond to the ring where they are drawn toappend.

The invention includes all pharmaceutically acceptable salt forms of thecompounds. Pharmaceutically acceptable salts are those in which thecounter ions do not contribute significantly to the physiologicalactivity or toxicity of the compounds and as such function aspharmacological equivalents. These salts can be made according to commonorganic techniques employing commercially available reagents. Someanionic salt forms include acetate, acistrate, besylate, bromide,camsylate, chloride, citrate, fumarate, glucouronate, hydrobromide,hydrochloride, hydroiodide, iodide, lactate, maleate, mesylate, nitrate,pamoate, phosphate, succinate, sulfate, tartrate, tosylate, andxinofoate. Some cationic salt forms include ammonium, aluminum,benzathine, bismuth, calcium, choline, diethylamine, diethanolamine,lithium, magnesium, meglumine, 4-phenylcyclohexylamine, piperazine,potassium, sodium, tromethamine, and zinc.

Some of the compounds of the invention possess asymmetric carbon atoms.The invention includes all stereoisomeric forms, including enantiomersand diastereomers as well as mixtures of stereoisomers such asracemates. Some stereoisomers can be made using methods known in theart. Stereoisomeric mixtures of the compounds and related intermediatescan be separated into individual isomers according to methods commonlyknown in the art. The use of wedges or hashes in the depictions ofmolecular structures in the following schemes and tables is intendedonly to indicate relative stereochemistry, and should not be interpretedas implying absolute stereochemical assignments.

The invention is intended to include all isotopes of atoms occurring inthe present compounds. Isotopes include those atoms having the sameatomic number but different mass numbers. By way of general example andwithout limitation, isotopes of hydrogen include deuterium and tritium.Isotopes of carbon include ¹³C and ¹⁴C. Isotopically-labeled compoundsof the invention can generally be prepared by conventional techniquesknown to those skilled in the art or by processes analogous to thosedescribed herein, using an appropriate isotopically-labeled reagent inplace of the non-labeled reagent otherwise employed. Such compounds mayhave a variety of potential uses, for example as standards and reagentsin determining biological activity. In the case of stable isotopes, suchcompounds may have the potential to favorably modify biological,pharmacological, or pharmacokinetic properties.

As set forth above, the invention is directed to one or more compoundsof Formula I, including pharmaceutically acceptable salts thereof:

wherein

-   m is 0 or 1;-   X is N or C—R¹⁰;-   R¹ is methyl;-   R² is Ar¹;-   Ar¹ is phenyl, 5-membered heteroaryl or 6-membered heteroaryl, and    is substituted with 0-3 substituents selected from the group of    cyano, halo, alkyl, cycloalkyl, haloalkyl, OH, OR¹⁰¹, haloalkoxy,    NH₂, NR¹⁰²R¹⁰³, COOR¹⁰¹, CONR¹⁰²R¹⁰³, S(O)₂R¹⁰¹, S(O)₂NR¹⁰²R¹⁰³, and    NR¹⁰¹CONR¹⁰²R¹⁰³;-   R¹⁰¹ is hydrogen, alkyl or cycloalkyl with 0-3 substituents selected    from halo, hydroxyl, alkoxy, and haloalkoxy;-   R¹⁰², R¹⁰³ are each independently hydrogen, alkyl or cycloalkyl with    0-3 substituents selected from halo, hydroxyl, alkoxy, and    haloalkoxy; or-   R¹⁰² and R¹⁰³ can form a ring by joining two atoms, one from each of    R¹⁰² and R¹⁰³;-   R³ is hydrogen, halo, or alkyl;-   R⁴, R⁵, R⁶, R⁷, R⁸ are each independently selected from the group of    hydrogen, halo, alkyl, cycloalkyl, haloalkyl, halocycloalkyl,    hydroxyalkyl, hydroxycycloalkyl, alkoxyalkyl, alkoxycycloalkyl,    alkoxy, hydroxyalkyloxy, alkoxyalkyloxy, COOR²⁰¹ and    CON(R²⁰²)(R²⁰³);-   R²⁰¹ is hydrogen, alkyl or cycloalkyl with 0-3 substituents selected    from halo, hydroxyl, alkoxy, and haloalkoxy;-   R²⁰², R²⁰³ are each independently selected from the group of    hydrogen, alkyl, cycloalkyl, alkenyl, cycloalkyl alkynyl, cyclic    ether, cyclic amine, lactame, fused bicyclic alkyl, bridged bicyclic    alkyl, spiro bicyclic alkyl, fused bicyclic ether, bridged bicyclic    ether, spiro bicyclic ether, fused bicyclic amine, bridged bicyclic    amine and spiro bicyclic amine, with 0-3 substituents selected from    the group of halo, OH, OR¹⁰⁴, NH₂, NR¹⁰⁵R¹⁰⁶, COOR¹⁰⁴, CONR¹⁰⁵R¹⁰⁶,    S(O)₂R¹⁰⁴, S(O)₂NR¹⁰⁵R¹⁰⁶, NR¹⁰⁴CONR¹⁰⁵R¹⁰⁶, OR¹⁰⁴CONR¹⁰⁵R¹⁰⁶,    C(═NR¹⁰⁷)NR¹⁰⁵R¹⁰⁶, NR¹⁰⁸C(═NR¹⁰⁷)NR¹⁰⁵R¹⁰⁶, haloalkoxy, Ar⁵, O—Ar²,    and NR¹⁰⁵—Ar²; or-   R²⁰² and R²⁰³ can form a ring by joining two atoms, one from each of    R²⁰² and R²⁰³;-   R²⁰² and R²⁰³ can also form bicyclic or tricyclic rings by joining    multiple atoms from each of R²⁰² and R²⁰³;-   R¹⁰⁴ is hydrogen, alkyl or cycloalkyl with 0-3 substituents selected    from halo, hydroxyl, alkoxy, and haloalkoxy;-   R¹⁰⁵, R¹⁰⁶ are each independently hydrogen, alkyl or cycloalkyl with    0-3 substituents selected from halo, hydroxyl, alkoxy, and    haloalkoxy; or-   R¹⁰⁵ and R¹⁰⁶ can form a ring by joining two atoms, one from each of    R¹⁰⁵ and R¹⁰⁶;-   R¹⁰⁷, R¹⁰⁸ are each independently hydrogen, alkyl or cycloalkyl with    0-3 substituents selected from halo, hydroxyl, alkoxy, and    haloalkoxy; or-   R¹⁰⁷ and R¹⁰⁸ can form a ring by joining two atoms, one from each of    R¹⁰⁷ and R¹⁰⁸;-   Ar² is phenyl, 5-membered heteroaryl or 6-membered heteroaryl, and    is substituted with 0-3 substituents selected from the group of    cyano, halo, alkyl, cycloalkyl, haloalkyl, OH, OR²⁰⁴, haloalkoxy,    NH₂, NR²⁰⁵R²⁰⁶, COOR²⁰⁴, CONR²⁰⁵R²⁰⁶, S(O)₂R²⁰⁴, S(O)₂NR²⁰⁵R²⁰⁶, and    NR²⁰⁴CONR²⁰⁵R²⁰⁶;-   R²⁰⁴ is hydrogen, alkyl or cycloalkyl with 0-3 substituents selected    from halo, hydroxyl, alkoxy, and haloalkoxy;-   R²⁰⁵, R²⁰⁶ are each independently hydrogen, alkyl or cycloalkyl with    0-3 substituents selected from halo, hydroxyl, alkoxy, and    haloalkoxy; or-   R²⁰⁵ and R²⁰⁶ can form a ring by joining two atoms, one from each of    R²⁰⁵ and R²⁰⁶;-   R⁹ is selected from the group of hydrogen, halo, alkyl, haloalkyl,    cycloalkyl, alkoxy, Ar³ and NR³⁰¹R³⁰²;-   R³⁰¹ is selected from the group of hydrogen, alkyl, cycloalkyl,    (cycloalkyl)alkyl, benzyl, alkylcarbonyl, haloalkyl carbonyl, phenyl    carbonyl, (alkoxyphenyl)carbonyl, alkylsulfonyl, phenylsulfonyl,    (alkoxyphenyl)sulfonyl and (haloalkoxyphenyl)sulfonyl;-   R³⁰² is hydrogen, alkyl, hydroxyalkyl, or alkoxyalkyl; or-   R³⁰¹ and R³⁰² taken together with the nitrogen to which they are    attached is oxazolidinonyl or dioxothiazinyl;-   Ar³ is phenyl, 5-membered heteroaryl or 6-membered heteroaryl, and    is substituted with 0-3 substituents selected from the group of    cyano, halo, alkyl, cycloalkyl, haloalkyl, OH, OR³⁰³, haloalkoxy,    NH₂, NR³⁰⁴R³⁰⁵, COOR³⁰³, CONR³⁰⁴R³⁰⁵, S(O)₂R³⁰³, S(O)₂NR³⁰⁴R³⁰⁵, and    NR³⁰³CONR³⁰⁴R³⁰⁵;-   R³⁰³ is hydrogen, alkyl or cycloalkyl with 0-3 substituents selected    from halo, hydroxyl, alkoxy, and haloalkoxy;-   R³⁰⁴, R³⁰⁵ are each independently hydrogen, alkyl or cycloalkyl with    0-3 substituents selected from halo, hydroxyl, alkoxy, and    haloalkoxy; or-   R³⁰⁴ and R³⁰⁵ can form a ring by joining two atoms, one from each of    R³⁰⁴ and R³⁰⁵;-   R¹⁰ is hydrogen, alkyl, halo, N(R⁴⁰¹)(R⁴⁰²), or alkylsulfonyl;-   R⁴⁰¹ and R⁴⁰² are independently selected from the group of hydrogen,    alkyl, hydroxyalkyl, alkoxyalkyl, alkylsulfonyl, and    alkylsulfonylalkyl;-   or N(R⁴⁰¹)(R⁴⁰²) taken together is azetidinyl, pyrrolidinyl,    piperidinyl, or piperazinyl, and is substituted with 0-2    substituents selected from alkyl, hydroxyalkyl, and hydroxy.

In a preferred embodiment of the invention, m is 0.

It is further preferred that R¹ is alkyl, and more preferably methyl.

It is also preferred that R⁴, R⁵, R⁶, R⁷, R⁸ are each selected fromCON(R²⁰²)(R²⁰³). More preferably, R⁵ is CON(R²⁰²)(R²⁰³).

Additionally, it is preferred that R²⁰² and R²⁰³ are hydrogen or alkyl.

In certain embodiments it is also preferred that R² is a 5-memberedheteroaryl group. More preferably, the heteroaryl group will contain 2to 3 nitrogens.

It is also preferred that R⁹ is selected from the group of haloalkyl,Ar³ and NR³⁰¹R³⁰². The haloalkyl is preferably 1-3 fluoroalkyl, and morepreferably is 1-3 fluoropropyl. In another embodiment, it is preferredthat R⁹ is NR³⁰¹, and R³⁰¹ is selected from alkyl and alkylsulfonyl. Ina further embodiment, it is preferred that Ar³ is substituted pyrazole.

R¹⁰ is preferably hydrogen.

Also preferred are compounds of Formula I, including pharmaceuticallyacceptable salts thereof, which are selected from the group of:

Also preferred are compounds of Formula I, including pharmaceuticallyacceptable salts thereof, which are selected from the group of:

Pharmaceutical Compositions and Methods of Treatment

The compounds according to the various embodiments herein set forthdemonstrate activity against HCV NS5B, and can be useful in treating HCVand HCV infection. Therefore, another aspect of the invention is acomposition comprising a compound of Formula I, or a pharmaceuticallyacceptable salt thereof, and a pharmaceutically acceptable carrier,excipient and/or diluent.

Another aspect of the invention is a composition further comprising anadditional compound having anti-HCV activity.

Another aspect of the invention is a composition where the compoundhaving anti-HCV activity is an interferon or a ribavirin. Another aspectof the invention is wherein the interferon is selected from interferonalpha 2B, pegylated interferon alpha, consensus interferon, interferonalpha 2A, interferon lambda, and lymphoblastoid interferon tau.

Another aspect of the invention is a composition where the compoundhaving anti-HCV activity is a cyclosporin. Another aspect of theinvention is where the cyclosporin is cyclosporin A.

Another aspect of the invention is a composition where the compoundhaving anti-HCV activity is selected from the group consisting ofinterleukin 2, interleukin 6, interleukin 12, a compound that enhancesthe development of a type 1 helper T cell response, interfering RNA,anti-sense RNA, Imiqimod, ribavirin, an inosine 5′-monophospatedehydrogenase inhibitor, amantadine, and rimantadine.

Another aspect of the invention is a composition where the compoundhaving anti-HCV activity is effective to inhibit the function of atarget selected from HCV metalloprotease, HCV serine protease, HCVpolymerase, HCV helicase, HCV NS4B protein, HCV entry, HCV assembly, HCVegress, HCV NS5A protein, IMPDH, and a nucleoside analog for thetreatment of an HCV infection.

Another aspect of the invention is a composition comprising a compoundof Formula I, or a pharmaceutically acceptable salt thereof, apharmaceutically acceptable carrier, an interferon and ribavirin.

Another aspect of the invention is a method of inhibiting the functionof the HCV replicon comprising contacting the HCV replicon with acompound of Formula I or a pharmaceutically acceptable salt thereof.

Another aspect of the invention is a method of inhibiting the functionof the HCV NS5B protein comprising contacting the HCV NS5B protein witha compound of Formula I or a pharmaceutically acceptable salt thereof.

Another aspect of the invention is a method of treating an HCV infectionin a patient comprising administering to the patient a therapeuticallyeffective amount of a compound of Formula I or a pharmaceuticallyacceptable salt thereof. In another embodiment the compound is effectiveto inhibit the function of the HCV replicon. In another embodiment thecompound is effective to inhibit the function of the HCV NS5B protein.

Another aspect of the invention is a method of treating an HCV infectionin a patient comprising administering to the patient a therapeuticallyeffective amount of a compound of Formula I, or a pharmaceuticallyacceptable salt thereof, in conjunction with (prior to, after, orconcurrently) another compound having anti-HCV activity.

Another aspect of the invention is the method wherein the other compoundhaving anti-HCV activity is an interferon or a ribavirin.

Another aspect of the invention is the method where the interferon isselected from interferon alpha 2B, pegylated interferon alpha, consensusinterferon, interferon alpha 2A, interferon lambda, and lymphoblastoidinterferon tau.

Another aspect of the invention is the method where the other compoundhaving anti-HCV activity is a cyclosporin.

Another aspect of the invention is the method where the cyclosporin iscyclosporin A.

Another aspect of the invention is the method where the other compoundhaving anti-HCV activity is selected from interleukin 2, interleukin 6,interleukin 12, a compound that enhances the development of a type 1helper T cell response, interfering RNA, anti-sense RNA, Imiqimod,ribavirin, an inosine 5′-monophospate dehydrogenase inhibitor,amantadine, and rimantadine.

Another aspect of the invention is the method wherein the other compoundhaving anti-HCV activity is effective to inhibit the function of atarget selected from the group consisting of HCV metalloprotease, HCVserine protease, HCV polymerase, HCV helicase, HCV NS4B protein, HCVentry, HCV assembly, HCV egress, HCV NS5A protein, IMPDH, and anucleoside analog for the treatment of an HCV infection.

Another aspect of the invention is the method wherein the other compoundhaving anti-HCV activity is effective to inhibit the function of targetin the HCV life cycle other than the HCV NS5B protein.

“Therapeutically effective” means the amount of agent required toprovide a meaningful patient benefit as understood by practitioners inthe field of hepatitis and HCV infection.

“Patient” means a person infected with the HCV virus and suitable fortherapy as understood by practitioners in the field of hepatitis and HCVinfection.

“Treatment,” “therapy,” “regimen,” “HCV infection,” and related termsare used as understood by practitioners in the field of hepatitis andHCV infection.

The compounds of this invention are generally given as pharmaceuticalcompositions comprised of a therapeutically effective amount of acompound or its pharmaceutically acceptable salt and a pharmaceuticallyacceptable carrier and may contain conventional excipients.Pharmaceutically acceptable carriers are those conventionally knowncarriers having acceptable safety profiles. Compositions encompass allcommon solid and liquid forms including for example capsules, tablets,lozenges, and powders as well as liquid suspensions, syrups, elixers,and solutions. Compositions are made using common formulationtechniques, and conventional excipients (such as binding and wettingagents) and vehicles (such as water and alcohols) are generally used forcompositions. See, for example, Remington's Pharmaceutical Sciences,Mack Publishing Company, Easton, Pa., 17th edition, 1985.

Solid compositions are normally formulated in dosage units andcompositions providing from about 1 to 1000 mg of the active ingredientper dose are preferred. Some examples of dosages are 1 mg, 10 mg, 100mg, 250 mg, 500 mg, and 1000 mg. Generally, other agents will be presentin a unit range similar to agents of that class used clinically.Typically, this is 0.25-1000 mg/unit.

Liquid compositions are usually in dosage unit ranges. Generally, theliquid composition will be in a unit dosage range of about 1-100 mg/mL.Some examples of dosages are 1 mg/mL, 10 mg/mL, 25 mg/mL, 50 mg/mL, and100 mg/mL. Generally, other agents will be present in a unit rangesimilar to agents of that class used clinically. Typically, this is1-100 mg/mL.

The invention encompasses all conventional modes of administration; oraland parenteral methods are preferred. Generally, the dosing regimen willbe similar to other agents used clinically. Typically, the daily dosewill be about 1-100 mg/kg body weight daily. Generally, more compound isrequired orally and less parenterally. The specific dosing regimen,however, will be determined by a physician using sound medical judgment.

The invention also encompasses methods where the compound is given incombination therapy. That is, the compound can be used in conjunctionwith, but separately from, other agents useful in treating hepatitis andHCV infection. In these combination methods, the compound will generallybe given in a daily dose of 1-100 mg/kg body weight daily in conjunctionwith other agents. The other agents generally will be given in theamounts used therapeutically. The specific dosing regimen, however, willbe determined by a physician using sound medical judgment.

Some examples of compounds suitable for compositions and methods as partof the invention herein are listed in Table 1.

TABLE 1 Type of Inhibitor or Brand Name Physiological Class TargetSource Company NIM811 Cyclophilin Inhibitor Novartis ZadaxinImmuno-modulator Sciclone Suvus Methylene blue Bioenvision Actilon TLR9agonist Coley (CPG10101) Batabulin (T67) Anticancer β-tubulin inhibitorTularik Inc., South San Francisco, CA ISIS 14803 Antiviral antisenseISIS Pharmaceuticals Inc, Carlsbad, CA/Elan Phamaceuticals Inc., NewYork, NY Summetrel Antiviral antiviral Endo Pharmaceuticals HoldingsInc., Chadds Ford, PA GS-9132 (ACH- Antiviral HCV InhibitorAchillion/Gilead 806) Pyrazolopyrimidine Antiviral HCV Inhibitors ArrowTherapeutics compounds and Ltd. salts From WO- 2005047288 26 May 2005Levovirin Antiviral IMPDH inhibitor Ribapharm Inc., Costa Mesa, CAMerimepodib Antiviral IMPDH inhibitor Vertex (VX-497) PharmaceuticalsInc., Cambridge, MA XTL-6865 (XTL- Antiviral monoclonal antibody XTL002) Biopharmaceuticals Ltd., Rehovot, Israel Telaprevir Antiviral NS3serine protease Vertex (VX-950, LY- inhibitor Pharmaceuticals 570310)Inc., Cambridge, MA/Eli Lilly and Co. Inc., Indianapolis, IN HCV-796Antiviral NS5B Replicase Wyeth/Viropharma Inhibitor NM-283 AntiviralNS5B Replicase Idenix/Novartis Inhibitor GL-59728 Antiviral NS5BReplicase Gene Labs/ Inhibitor Novartis GL-60667 Antiviral NS5BReplicase Gene Labs/ Inhibitor Novartis 2′C MeA Antiviral NS5B ReplicaseGilead Inhibitor PSI 6130 Antiviral NS5B Replicase Roche Inhibitor R1626Antiviral NS5B Replicase Roche Inhibitor 2′C Methyl Antiviral NS5BReplicase Merck adenosine Inhibitor JTK-003 Antiviral RdRp inhibitorJapan Tobacco Inc., Tokyo, Japan Levovirin Antiviral ribavirin ICNPharmaceuticals, Costa Mesa, CA Ribavirin Antiviral ribavirinSchering-Plough Corporation, Kenilworth, NJ Viramidine AntiviralRibavirin Prodrug Ribapharm Inc., Costa Mesa, CA Heptazyme Antiviralribozyme Ribozyme Pharmaceuticals Inc., Boulder, CO BILN-2061 Antiviralserine protease Boehringer inhibitor Ingelheim Pharma KG, Ingelheim,Germany SCH 503034 Antiviral serine protease Schering Plough inhibitorZadazim Immune modulator Immune modulator SciClone Pharmaceuticals Inc.,San Mateo, CA Ceplene Immunomodulator immune modulator MaximPharmaceuticals Inc., San Diego, CA CellCept Immunosuppressant HCV IgGimmuno- F. Hoffmann-La suppressant Roche LTD, Basel, Switzerland CivacirImmunosuppressant HCV IgG immuno- Nabi suppressant BiopharmaceuticalsInc., Boca Raton, FL Albuferon-α Interferon albumin IFN-α2b Human GenomeSciences Inc., Rockville, MD Infergen A Interferon IFN InterMunealfacon-1 Pharmaceuticals Inc., Brisbane, CA Omega IFN Interferon IFN-ωIntarcia Therapeutics IFN-β and EMZ701 Interferon IFN-β and EMZ701Transition Therapeutics Inc., Ontario, Canada Rebif Interferon IFN-β1aSerono, Geneva, Switzerland Roferon A Interferon IFN-α2a F. Hoffmann-LaRoche LTD, Basel, Switzerland Intron A Interferon IFN-α2bSchering-Plough Corporation, Kenilworth, NJ Intron A and InterferonIFN-α2b/α1-thymosin RegeneRx Zadaxin Biopharma. Inc., Bethesda, MD/SciClone Pharmaceuticals Inc, San Mateo, CA Rebetron InterferonIFN-α2b/ribavirin Schering-Plough Corporation, Kenilworth, NJ ActimmuneInterferon INF-γ InterMune Inc., Brisbane, CA Interferon-β InterferonInterferon-β-1a Serono Multiferon Interferon Long lasting IFN Viragen/Valentis Wellferon Interferon Lympho-blastoid IFN- GlaxoSmithKline αn1plc, Uxbridge, UK Omniferon Interferon natural IFN-α Viragen Inc.,Plantation, FL Pegasys Interferon PEGylated IFN-α2a F. Hoffmann-La RocheLTD, Basel, Switzerland Pegasys and Interferon PEGylated IFN-α2a/ MaximCeplene immune modulator Pharmaceuticals Inc., San Diego, CA Pegasys andInterferon PEGylated IFN- F. Hoffmann-La Ribavirin α2a/ribavirin RocheLTD, Basel, Switzerland PEG-Intron Interferon PEGylated IFN-α2bSchering-Plough Corporation, Kenilworth, NJ PEG-Intron/ InterferonPEGylated IFN- Schering-Plough Ribavirin α2b/ribavirin Corporation,Kenilworth, NJ IP-501 Liver protection antifibrotic IndevusPharmaceuticals Inc., Lexington, MA IDN-6556 Liver protection caspaseinhibitor Idun Pharmaceuticals Inc., San Diego, CA ITMN-191 (R-7227)Antiviral serine protease InterMune inhibitor Pharmaceuticals Inc.,Brisbane, CA GL-59728 Antiviral NS5B Replicase Genelabs InhibitorANA-971 Antiviral TLR-7 agonist Anadys Boceprevir Antiviral serineprotease Schering Plough inhibitor TMS-435 Antiviral serine proteaseTibotec BVBA, inhibitor Mechelen, Belgium BI-201335 Antiviral serineprotease Boehringer inhibitor Ingelheim Pharma KG, Ingelheim, GermanyMK-7009 Antiviral serine protease Merck inhibitor PF-00868554 Antiviralreplicase inhibitor Pfizer ANA598 Antiviral Non-Nucleoside Anadys NS5BPolymerase Pharmaceuticals, Inhibitor Inc., San Diego, CA, USA IDX375Antiviral Non-Nucleoside Idenix Replicase Inhibitor Pharmaceuticals,Cambridge, MA, USA BILB 1941 Antiviral NS5B Polymerase BoehringerInhibitor Ingelheim Canada Ltd R&D, Laval, QC, Canada PSI-7851 AntiviralNucleoside Pharmasset, Polymerase Inhibitor Princeton, NJ, USA PSI-7977Antiviral Nucleotide NS5B Pharmasset, Polymerase Inhibitor Princeton,NJ, USA VCH-759 Antiviral NS5B Polymerase ViroChem Pharma InhibitorVCH-916 Antiviral NS5B Polymerase ViroChem Pharma Inhibitor GS-9190Antiviral NS5B Polymerase Gilead Inhibitor Peg-interferon AntiviralInterferon ZymoGenetics/Bristol- lambda Myers Squibb

Synthesis Methods

The compounds may be made by methods available in the art, as well asthose described below. Some reagents and intermediates are available inthe art. Other reagents and intermediates can be made by methodsavailable in the art using commercially available materials. Thevariables (e.g. numbered “R” substituents) used to describe thesynthesis of the compounds are intended only to illustrate how to makethese compounds, and are not to be confused with variables used in theclaims or in other sections of the specification. Abbreviations usedwithin the schemes generally follow conventions used in the art.

Abbreviations used in the schemes generally follow conventions used inthe art. Chemical abbreviations used in the specification and examplesare defined as follows: “NaHMDS” for sodium bis(trimethylsilyl)amide;“DMF” for N,N-dimethylformamide; “MeOH” for methanol; “NBS” forN-bromosuccinimide; “Ar” for aryl; “TFA” for trifluoroacetic acid; “LAH”for lithium aluminum hydride; “DMSO” for dimethylsulfoxide; “h” forhours; “rt” for room temperature or retention time (context willdictate); “min” for minutes; “EtOAc” for ethyl acetate; “THF” fortetrahydrofuran; “EDTA” for ethylenediaminetetraacetic acid; “Et₂O” fordiethyl ether; “DMAP” for 4-dimethylaminopyridine; “DCE” for1,2-dichloroethane; “ACN” for acetonitrile; “DME” for1,2-dimethoxyethane; “HOBt” for 1-hydroxybenzotriazole hydrate; “DIEA”for diisopropylethylamine.

For the section of compounds in the 0000 series all LiquidChromatography (LC) data were recorded on a Shimadzu LC-10AS or LC-20ASliquid chromotograph using a SPD-10AV or SPD-20A UV-Vis detector andMass Spectrometry (MS) data were determined with a Micromass Platformfor LC in electrospray mode.

HPLC Method (i.e., compound isolation). Compounds purified bypreparative HPLC were diluted in methanol (1.2 mL) and purified using aShimadzu LC-8A or LC-10A or Dionex APS-3000 or Waters Acquity™ automatedpreparative HPLC system.

EXAMPLES

Preparation of Compound 1001:

A mixture of 1H-pyrazole (54.2 mg),5-(3-(tert-butylcarbamoyl)phenyl)-6-(N-ethylmethylsulfonamido)-2-(4-fluorophenyl)-N-methylbenzofuran-3-carboxamide(15 mg), K₂CO₃ (14.66 mg) and copper (3.37 mg) was heated to 140° C. for6 hours. The mixture was diluted with DMF and filtered to removed solid.The solution of mixture was subjected to purification by preparativeHPLC system.

1001 MS (M + H)⁺ Calcd. 614.2 MS (M + H)⁺ Observ. 614.2 Retention Time1.35 min LC Condition Solvent A 90% Water-10% Methanol-0.1% TFA SolventB 10% Water-90% Methanol-0.1% TFA Start % B  50 Final % B 100 GradientTime 2 min Flow Rate 1 mL/min Wavelength 220 Solvent PairWater-Methanol-TFA Column PHENOMENEX-LUNA 2.0 × 30 mm 3 umPreparation of Compounds 1002 and 1003:

Compounds 1002 and 1003 were prepared via the same procedure towardscompound 1001, using 1,2,3-triazole. LC condition for compounds 1002 and1003 was the same for compound 100.

1002 MS (M + H)⁺ Calcd. 615.2 MS (M + H)⁺ Observ. 615.2 Retention Time1.18 min

1003 MS (M + H)⁺ Calcd. 615.2 MS (M + H)⁺ Observ. 615.2 Retention Time1.20 minPreparation of Compound 2001:

NaH (8.33 mg, 60%) was added into a solution of5-(3-(tert-butylcarbamoyl)phenyl)-6-chloro-2-(4-fluorophenyl)-N-methylfuro[2,3-b]pyridine-3-carboxamide(10 mg) and 3-(trifluoromethyl)-1H-pyrazole (28.4 mg) in DMF. Thereaction was heated at 85° C. for 24 hour. The product was isolated bypreparative HPLC system.

2001 MS (M + H)⁺ Calcd. 696.2 MS (M + H)⁺ Observ. 696.2 Retention Time2.38 min LC Condition Solvent A 90% Water-10% Methanol-0.1% TFA SolventB 10% Water-90% Methanol-0.1% TFA Start % B  0 Final % B 100 GradientTime 2 min Flow Rate 1 mL/min Wavelength 220 Solvent PairWater-Methanol-TFA Column PHENOMENEX-LUNA 2.0 × 30 mm 3 umPreparation of Compound 2002:Compound 2002 were prepared via the same procedure towards compound2001, using 4-(trifluoromethyl)-1H-pyrazole. LC condition for compound2002 was the same for compound 2001.

2002

MS (M + H)⁺ Calcd. 696.2 MS (M + H)⁺ Observ. 696.2 Retention Time  2.40minPreparation of Compound 3001:

A mixture of 1H-1,2,3-triazole (96 mg),5-(3-(tert-butylcarbamoyl)phenyl)-2-(4-fluorophenyl)-N-methyl-6-(3,3,3-trifluoropropyl)furo[2,3-b]pyridine-3-carboxamide(15 mg), K₂CO₃ (11.48 mg) and copper (3.52 mg) was heated to 140° C. for6 hours. The mixture was diluted with DMF and filtered to removed solid.The solution of mixture was subjected to purification by preparativeHPLC system.

3001 MS (M + H)⁺ Calcd. 591.2 MS (M + H)⁺ Observ. 591.2 Retention Time1.63 min LC Condition Solvent A 90% Water-10% Methanol-0.1% TFA SolventB 10% Water-90% Methanol-0.1% TFA Start % B  50 Final % B 100 GradientTime 2 min Flow Rate 1 mL/min Wavelength 220 Solvent PairWater-Methanol-TFA Column PHENOMENEX-LUNA 2.0 × 30 mm 3 umCompounds 3002-3010 were prepared via the same procedure towardscompound 3001, using the corresponding heterocycles. LC condition forcompounds 3002-3010 was the same for compound 3001.

MS MS Retention (M + H)⁺ (M + H)⁺ Time Cmpd # Structure Calcd. Observ.(min) 3002

605.2 605.2 1.31 3003

605.2 605.2 1.58 3004

605.2 605.2 1.73 3005

590.2 590.2 1.79 3006

590.2 590.2 1.08 3007

591.2 591.2 1.64 3008

604.3 604.2 1.93 3009

604.3 604.2 1.17 3010

604.3 604.2 1.89Preparation of Compound 4001:Compound 4001 was prepared via the same procedure towards compound 3001,using pyridin-3-ol. LC condition for compound 4001 was the same forcompound 3001.

4001

MS (M + H)⁺ Calcd. 617.2 MS (M + H)⁺ Observ. 617.2 Retention Time  1.10min LC Condition Solvent A  90% Water −10% Methanol-0.1% TFA Solvent B 10% Water −90% Methanol-0.1% TFA Start % B  50 Final % B 100 GradientTime  2 min Flow Rate  1 mL/min Wavelength 220 Solvent Pair Water -Methanol-TFA Column PHENOMENEX-LUNA 2.0 × 30 mm 3 um

Biological Methods

The compound demonstrated activity against HCV NS5B as determined in thefollowing HCV RdRp assays.

HCV NS5B RdRp cloning, expression, and purification. The cDNA encodingNS5B proteins of HCV genotype 1b (Con1), a genotype 1b variant withamino acid 316 mutated from cysteine to asparagine, and genotype 2a(JFH-1), were cloned into the pET21a expression vector. Each untaggedprotein was expressed with an 18 amino acid C-terminal truncation toenhance the solubility. The E. coli competent cell line BL21(DE3) wasused for expression of the protein. Cultures were grown at 37° C. for ˜4hours until the cultures reached an optical density of 2.0 at 600 nm.The cultures were cooled to 20° C. and induced with 1 mM IPTG. Freshampicillin was added to a final concentration of 50 μg/mL and the cellswere grown overnight at 20° C.

Cell pellets (3 L) were lysed for purification to yield 15-24 mgs ofpurified NS5B. The lysis buffer consisted of 20 mM Tris-HC1, pH 7.4, 500mM NaCl, 0.5% triton X-100, 1 mM DTT, 1 mM EDTA, 20% glycerol, 0.5 mg/mLlysozyme, 10 mM MgCl₂, 15 ug/mL deoxyribonuclease I, and Complete TMprotease inhibitor tablets (Roche). After addition of the lysis buffer,frozen cell pellets were resuspended using a tissue homogenizer. Toreduce the viscosity of the sample, aliquots of the lysate weresonicated on ice using a microtip attached to a Branson sonicator. Thesonicated lysate was centrifuged at 100,000×g for 30 minutes at 4° C.and filtered through a 0.2 μm filter unit (Corning).

The protein was purified using two sequential chromatography steps:Heparin sepharose CL-6B and polyU sepharose 4B. The chromatographybuffers were identical to the lysis buffer but contained no lysozyme,deoxyribonuclease I, MgCl₂ or protease inhibitor and the NaClconcentration of the buffer was adjusted according to the requirementsfor charging the protein onto the column. Each column was eluted with aNaCl gradient which varied in length from 5-50 column volumes dependingon the column type. After the final chromatography step, the resultingpurity of the enzyme is >90% based on SDS-PAGE analysis. The enzyme wasaliquoted and stored at −80° C.

HCV NS5B RdRp enzyme assay. An on-bead solid phase homogeneous assay wasused in a 384-well format to assess NS5B inhibitors (WangY-K, Rigat K,Roberts S, and Gao M (2006) Anal Biochem, 359: 106-111). Thebiotinylated oligo dT₁₂ primer was captured on streptavidin-coupledimaging beads (GE, RPNQ0261) by mixing primer and beads in 1× buffer andincubating at room temperature for three hours. Unbound primer wasremoved after centrifugation. The primer-bound beads were resuspended in3× reaction mix (20 mM Hepes buffer, pH 7.5, dT primer coupled beads,poly A template, ³H-UTP, and RNAse inhibitor (Promega N2515)). Compoundswere serially diluted 1:3 in DMSO and aliquoted into assay plates. Equalvolumes (5 μL) of water, 3× reaction mix, and enzyme in 3× assay buffer(60 mM Hepes buffer, pH 7.5, 7.5 mM MgCl₂, 7.5 mM KCl, 3 mM DTT, 0.03mg/mL BSA, 6% glycerol) were added to the diluted compound on the assayplate. Final concentration of components in 384-well assay: 0.36 nMtemplate, 15 nM primer, 0.29 μM³H-UTP (0.3 μCi), 1.6 U/μL RNAseinhibitor, 7 nM NS5B enzyme, 0.01 mg/mL BSA, 1 mM DTT, and 0.33 μg/μLbeads, 20 mM Hepes buffer, pH 7.5, 2.5 mM MgCl₂, 2.5 mM KCl, and 0.1%DMSO.

Reactions were allowed to proceed for 24 hours at 30° C. and terminatedby the addition of 50 mM EDTA (5 μL). After incubating for at least 15minutes, plates were read on an Amersham LEADseeker multimodalityimaging system.

IC₅₀ values for compounds were determined using ten different [I]. IC₅₀values were calculated from the inhibition using the four-parameterlogistic formula y=A+ ((B−A)/(1+((C/x)^D))), where A and B denoteminimal and maximal % inhibition, respectively, C is the IC₅₀, D is hillslope and x represents compound concentration.

Cell lines. The cell lines used to evaluate compounds consist of a humanhepatocyte derived cell line (Huh-7) that constitutively expresses agenotype 1b (Con-1) HCV replicon or a genotype 1b (Con-1) HCV repliconwith an asparagine replacing the cysteine at amino acid 316, or agenotype 2a (JFH-1) replicon, containing a Renilla luciferase reportergene. These cells were maintained in Dulbecco's modified Eagle medium(DMEM) containing 10% FBS, 100 U/mL penicillin/streptomycin and 1.0mg/mL G418.

HCV replicon luciferase assay. To evaluate compound efficacy, titratedcompounds were transferred to sterile 384-well tissue culture treatedplates, and the plates were seeded with HCV replicon cells (50 μL at adensity of 2.4×10³ cells/well) in DMEM containing 4% FBS (final DMSOconcentration at 0.5%). After 3 days incubation at 37° C., cells wereanalyzed for Renilla Luciferase activity using the EnduRen substrate(Promega cat #E6485) according to the manufacturer's directions.Briefly, the EnduRen substrate was diluted in DMEM and then added to theplates to a final concentration of 7.5 μM. The plates were incubated forat least 1 h at 37° C. then read on a Viewlux Imager (PerkinElmer) usinga luminescence program. The 50% effective concentration (EC₅₀) wascalculated using the four-parameter logistic formula noted above.

To assess cytotoxicity of compounds, Cell Titer-Blue (Promega) was addedto the EnduRen-containing plates and incubated for at least 4 hrs at 37°C. The fluorescence signal from each well was read using a ViewluxImager. All CC₅₀ values were calculated using the four-parameterlogistic formula.

Compound EC₅₀ data is expressed as A:<100 nM; B=100-1000 nM; C>1000 nM).Representative data for compounds are reported in Table 2.

TABLE 2 EC₅₀ (uM) Cmpd # Structure lb 1001

0.017 A 1002

0.069 A 1003

A 2001

B 2002

C 3001

0.035 A 3002

C 3003

3.32  C 3004

B 3006

B 3007

B 3008

C 3009

C 3010

0.28  B 4001

B

It will be evident to one skilled in the art that the present disclosureis not limited to the foregoing illustrative examples, and that it canbe embodied in other specific forms without departing from the essentialattributes thereof. It is therefore desired that the examples beconsidered in all respects as illustrative and not restrictive,reference being made to the appended claims, rather than to theforegoing examples, and all changes which come within the meaning andrange of equivalency of the claims are therefore intended to be embracedtherein.

We claim:
 1. A compound of Formula I, including pharmaceuticallyacceptable salts thereof:

wherein m is 0 or 1; X is N or C—R¹⁰; R¹ is methyl; R² is Ar¹; Ar¹ isphenyl, 5-membered heteroaryl or 6-membered heteroaryl, and issubstituted with 0-3 substituents selected from the group of cyano,halo, alkyl, cycloalkyl, haloalkyl, OH, OR¹⁰¹, haloalkoxy, NH₂,NR¹⁰²R¹⁰³, COOR¹⁰¹, CONR¹⁰²R¹⁰³, S(O)₂R¹⁰¹, S(O)₂NR¹⁰²R¹⁰³, andNR¹⁰¹CONR¹⁰²R¹⁰³; R¹⁰¹ is hydrogen, alkyl or cycloalkyl with 0-3substituents selected from halo, hydroxyl, alkoxy, and haloalkoxy; R¹⁰²,R¹⁰³ are each independently hydrogen, alkyl or cycloalkyl with 0-3substituents selected from halo, hydroxyl, alkoxy, and haloalkoxy; orR¹⁰² and R¹⁰³ can form a ring by joining two atoms, one from each ofR¹⁰² and R¹⁰³; R³ is hydrogen, halo, or alkyl; R⁴, R⁵, R⁶, R⁷, R⁸ areeach independently selected from the group of hydrogen, halo, alkyl,cycloalkyl, haloalkyl, halocycloalkyl, hydroxyalkyl, hydroxycycloalkyl,alkoxyalkyl, alkoxycycloalkyl, alkoxy, hydroxyalkyloxy, alkoxyalkyloxy,COOR²⁰¹ and CON(R²⁰²)(R²⁰³); R²⁰¹ is hydrogen, alkyl or cycloalkyl with0-3 substituents selected from halo, hydroxyl, alkoxy, and haloalkoxy;R²⁰², R²⁰³ are each independently selected from the group of hydrogen,alkyl, cycloalkyl, alkenyl, cycloalkyl alkynyl, cyclic ether, cyclicamine, lactame, fused bicyclic alkyl, bridged bicyclic alkyl, spirobicyclic alkyl, fused bicyclic ether, bridged bicyclic ether, spirobicyclic ether, fused bicyclic amine, bridged bicyclic amine, and spirobicyclic amine, with 0-3 substituents selected from the group of halo,OH, OR¹⁰⁴, NH₂, NR¹⁰⁵R¹⁰⁶, COOR¹⁰⁴, CONR¹⁰⁵R¹⁰⁶, S(O)₂R¹⁰⁴,S(O)₂NR¹⁰⁵R¹⁰⁶, NR¹⁰⁴CONR¹⁰⁵R¹⁰⁶, OR¹⁰⁴CONR¹⁰⁵R¹⁰⁶, C(═NR¹⁰⁷)NR¹⁰⁵R¹⁰⁶,NR¹⁰⁸C(═NR¹⁰⁷)NR¹⁰⁵R¹⁰⁶, haloalkoxy, Ar², O—Ar², and NR¹⁰⁵—Ar²; or R²⁰²and R²⁰³ can form a ring by joining two atoms, one from each of R²⁰² andR²⁰³; R²⁰² and R²⁰³ can also form bicyclic or tricyclic rings by joiningmultiple atoms from each of R²⁰² and R²⁰³; R¹⁰⁴ is hydrogen, alkyl orcycloalkyl with 0-3 substituents selected from halo, hydroxyl, alkoxy,and haloalkoxy; R¹⁰⁵, R¹⁰⁶ are each independently hydrogen, alkyl orcycloalkyl with 0-3 substituents selected from halo, hydroxyl, alkoxy,and haloalkoxy; or R¹⁰⁵ and R¹⁰⁶ can form a ring by joining two atoms,one from each of R¹⁰⁵ and R¹⁰⁶; R¹⁰⁷, R¹⁰⁸ are each independentlyhydrogen, alkyl or cycloalkyl with 0-3 substituents selected from halo,hydroxyl, alkoxy, and haloalkoxy; or R¹⁰⁷ and R¹⁰⁸ can form a ring byjoining two atoms, one from each of R¹⁰⁷ and R¹⁰⁸; Ar² is phenyl,5-membered heteroaryl or 6-membered heteroaryl, and is substituted with0-3 substituents selected from the group of cyano, halo, alkyl,cycloalkyl, haloalkyl, OH, OR²⁰⁴, haloalkoxy, NH₂, NR²⁰⁵R²⁰⁶, COOR²⁰⁴,CONR²⁰⁵R²⁰⁶, S(O)₂R²⁰⁴, S(O)₂NR²⁰⁵R²⁰⁶, and NR²⁰⁴CONR²⁰⁵R²⁰⁶; R²⁰⁴ ishydrogen, alkyl or cycloalkyl with 0-3 substituents selected from halo,hydroxyl, alkoxy, and haloalkoxy; R²⁰⁵, R²⁰⁶ are each independentlyhydrogen, alkyl or cycloalkyl with 0-3 substituents selected from halo,hydroxyl, alkoxy, and haloalkoxy; or R²⁰⁵ and R²⁰⁶ can form a ring byjoining two atoms, one from each of R²⁰⁵ and R²⁰⁶; R⁹ is selected fromthe group of hydrogen, halo, alkyl, cycloalkyl, alkoxy, Ar³ andNR³⁰¹R³⁰²; R³⁰¹ is selected from the group of hydrogen, alkyl,cycloalkyl, (cycloalkyl)alkyl, benzyl, alkylcarbonyl, haloalkylcarbonyl, phenyl carbonyl, (alkoxyphenyl)carbonyl, alkylsulfonyl,phenylsulfonyl, (alkoxyphenyl)sulfonyl, and (haloalkoxyphenyl)sulfonyl;R³⁰² is hydrogen, alkyl, hydroxyalkyl, or alkoxyalkyl; or R³⁰¹ and R³⁰²taken together with the nitrogen to which they are attached isoxazolidinonyl or dioxothiazinyl; Ar³ is phenyl, 5-membered heteroarylor 6-membered heteroaryl, and is substituted with 0-3 substituentsselected from the group of cyano, halo, alkyl, cycloalkyl, haloalkyl,OH, OR³⁰³, haloalkoxy, NH₂, NR³⁰⁴R³⁰⁵, COOR³⁰³, CONR³⁰⁴R³⁰⁵, S(O)₂R³⁰³,S(O)₂NR³⁰⁴R³⁰⁵, and NR³⁰³CONR³⁰⁴R³⁰⁵; R³⁰³ is hydrogen, alkyl orcycloalkyl with 0-3 substituents selected from halo, hydroxyl, alkoxy,and haloalkoxy; R³⁰⁴, R³⁰⁵ are each independently hydrogen, alkyl orcycloalkyl with 0-3 substituents selected from halo, hydroxyl, alkoxy,and haloalkoxy; or R³⁰⁴ and R³⁰⁵ can form a ring by joining two atoms,one from each of R³⁰⁴ and R³⁰⁵; R¹⁰ is hydrogen, alkyl, halo,N(R⁴⁰¹)(R⁴⁰²), or alkylsulfonyl; R⁴⁰¹ and R⁴⁰² are each independentlyselected from the group of hydrogen, alkyl, hydroxyalkyl, alkoxyalkyl,alkylsulfonyl, or alkylsulfonylalkyl; or N(R⁴⁰¹)(R⁴⁰²) taken together isazetidinyl, pyrrolidinyl, piperidinyl, or piperazinyl, and issubstituted with 0-2 substituents selected from alkyl, hydroxyalkyl, andhydroxy.
 2. A compound of claim 1 wherein m=0.
 3. A compound of claim 2wherein R¹ is alkyl.
 4. A compound of claim 3 wherein R⁵ isCON(R²⁰²)(R²⁰³).
 5. A compound of claim 1 wherein R²⁰² and R²⁰³ arehydrogen or alkyl.
 6. A compound of claim 1 wherein R² is a 5-memberedheteroaryl group.
 7. A compound of claim 6 wherein R² has 2 to 3nitrogens.
 8. A compound of claim 1 wherein R⁹ is selected from thegroup of haloalkyl, NR³⁰¹, R³⁰² and Ar³.
 9. A compound of claim 8wherein haloalkyl is 1-3 fluoroalkyl.
 10. A compound of claim 9 whereinsaid alkyl is propyl.
 11. A compound of claim 1 wherein R¹⁰ is hydrogen.12. A compound, including pharmaceutically acceptable salts thereof,which is selected from the group of:


13. A compound, including pharmaceutically acceptable salts thereof,which is selected from the group of:


14. A composition comprising a compound of claim 1 or a pharmaceuticallyacceptable salt thereof and a pharmaceutically acceptable carrier,excipient, and/or diluent.
 15. A composition comprising a compound ofclaim 12 or a pharmaceutically acceptable salt thereof and apharmaceutically acceptable carrier, excipient, and/or diluent.
 16. Acomposition comprising a compound of claim 13 or a pharmaceuticallyacceptable salt thereof and a pharmaceutically acceptable carrier,excipient, and/or diluent.
 17. A compound as claimed in claim 8, whereinR⁹ is NR³⁰¹, and R³⁰¹ is selected from alkyl and alkylsulfonyl.